Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, 100871, China.
Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, 100871, China.
Arch Biochem Biophys. 2021 Oct 30;711:109023. doi: 10.1016/j.abb.2021.109023. Epub 2021 Sep 1.
In eukaryotes, N6-methyladenosine (mA) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most mA researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of mA in small RNAs. Nevertheless, current knowledge about mA prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of mA methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific mA profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA mA immunoprecipitation sequencing and constructed the consensus motif in mA-enriched miRNAs de novo. We found that miR-21-5p, an oncogenic miRNA, showed the highest mA enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21-5p, but altered the mA abundance of miR-21-5p, thereby changing the expression levels of its target gene. We further synthesized mA modified miR-21-5p mimics in vitro and demonstrated that in NSCLC cells, mA marks in mature miR-21-5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of mA modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.
在真核生物中，N6-甲基腺嘌呤（m6A）是 RNA 上最丰富的修饰之一，它在许多生物过程和疾病中发挥着重要作用，如癌症。虽然大多数 mA 研究集中在信使 RNA 和长非编码 RNA 上，但最近的研究报道了 mA 在小 RNA 中的存在。然而，目前关于成熟 microRNA（miRNA）中 mA 普遍性的知识极其有限，m6A 甲基化在 miRNA 中的功能意义仍有待阐明。在这里，我们通过使用 miRNA m6A 免疫沉淀测序，在 A549 人非小细胞肺癌（NSCLC）细胞和 HEK293A 细胞中展示了 miRNA 的细胞特异性 m6A 图谱，并从头构建了 m6A 富集 miRNA 中的共识基序。我们发现，致癌 miRNA miR-21-5p 在 NSCLC 细胞中表现出最高的 m6A 富集。去甲基酶 ALKBH5 的耗竭不会改变 miR-21-5p 的表达水平，但会改变 miR-21-5p 的 m6A 丰度，从而改变其靶基因的表达水平。我们进一步在体外合成了 m6A 修饰的 miR-21-5p 模拟物，并证明在 NSCLC 细胞中，成熟 miR-21-5p 中的 m6A 标记可以直接影响其对靶基因的沉默能力，从而最终损害其对增殖和迁移的促进作用。总之，我们的研究结果揭示了成熟 miRNA 中 m6A 修饰的全景，并提供了第一个证据，表明它可能有助于癌症相关 miRNA 对 mRNA 的反应。
